The primary goal of this research is to explore the role of the nitric oxide/cGMP/protein kinase G in protecting hepatocytes from mitochondrial permeability transition (MPT)-dependent death after ischemia/reperfusion injury to hepatocytes. To achieve this aim, MPT assays with isolated rat liver mitochondria incubated with liver cytosol will be employed to assess the involvement of PKG and PKA pathways in regulating onset of the MPT. Viability assays and hepatocytes exposed to TNFa will be used to confirm results obtained from the MPT-assays with isolated mitochondria. As relevant kinases are identified, the second aim will to identify the mitochondrial proteins where kinase-dependent phosphorylation inhibits MPT onset. To achieve this aim, isolated mitochondria will be used in the presence of purified kinases. The proteins phosphorylated will be identified by 2-D electrophoresis and/or HPLC in conjunction with protein sequencing. Phospho-mapping techniques will be used to identify the discrete phosphorylation sites of the isolated proteins. These experiments will provide new information on regulation of the MPT by kinases, for targets of phosphorylation, and relevance of the kinases to MPT-dependent cell death. [unreadable] [unreadable]